1. Field of the Invention
The invention relates generally to the exportation and localization of polypeptides to the external membrane surface of a gram-negative cell, to recombinant vectors useful for the transformation of a host cell and to chimeric genes that provide outer membrane targeting and transmembrane sequences. Methods are disclosed providing for surface expression of proteins, including antigenically active proteins, specific binding proteins and enzymatically active species.
2. Description of Related Art
There is substantial interest in the expression of selected proteins on the surface of bacteria. Many potential applications exist, including the production of genetically engineered whole cell adsorbents, construction of "peptide libraries" where bacteria carry different exposed sequences, cell-bound enzymes (another form of immobilization), and use as live vaccines or immunogens to generate antibodies.
One approach to obtaining surface expressed foreign proteins has been to use a native membrane protein as a carrier for a foreign protein. LamB, an outer membrane protein of Escherichia coli, has been fused with peptides varying in length up to about 60 amino acids with successful expression of the hybrid protein at a recombinant host cell surface (Charbit, et al., 1991). Unfortunately, only relatively short polypeptides are surface-expressed using this method. Outer membrane proteins have "loop" regions spanning the membrane surface and while it is possible to substitute foreign DNA into the gene regions encoding the loop regions, there are only a limited number of insertions possible, constrained by the size of the loop region and, apparently, by the requirement to preserve the penetration and translocating properties of the membrane protein.
In general, attempts to develop methods of anchoring larger proteins as well as the smaller peptides on a bacterial cell surface have focused on fusion of the desired recombinant polypeptide to a native protein that is normally exposed on the cell's exterior in hope that the resulting hybrid will also be localized on the surface. The problem with this approach is that fusion of the foreign protein interferes with localization and, in many cases, the hybrid molecule is unable to reach the cell surface.
Nevertheless, in one example employing the Klebsiella enzyme pullulanase, a normally periplasmic protein, .beta.-lactamase, was translocated through the E. coli outer membrane. C-terminal regions of pullulanase were replaced with DNA segments encoding .beta.-lactamase or alkaline phosphatase. Only the hybrid protein with .beta.-lactamase was transported to the cell surface (Kornacker and Pugsley, 1990). However, the surface-expressed protein was only transiently anchored to the cell surface, suggesting a severe limitation on the potential value of any other proteins expressed by this method as surface immunogens, adsorbents, or surface immobilized species. Furthermore, the assembly of pullulanase fusions onto the cell surface is a very complicated process requiring the presence of at least 14 foreign gene products in the host cell. It should be noted that alkaline phosphatase fused to the same pullulanase sequence could not be localized on the cell surface (Kornacker and Pugsley 1990).
The mechanisms of protein insertion within- and translocation across- the outer membrane of gram-negative bacteria are not well understood. For some outer membrane proteins, such as the PhoE porin, the information necessary for proper localization and assembly is interspersed within the primary sequence (Bosch et al., 1986; Bosch et al., 1989). Alternatively, the targeting signal may be contained within a single short continuous segment. For example, the first nine N-terminal amino acids of the major E. coli lipoprotein are necessary for proper localization in the outer membrane. Fusion to this short sequence is sufficient to direct the normally soluble periplasmic protein .beta.-lactamase to the outer membrane (Ghrayeb and Inouye, 1984). Similarly, extensive studies with OmpA have suggested that the region between residues 154 and 180 is crucial for localization (Klose et al., 1988a, 1988b). With OmpA, targeting and outer membrane assembly appear to be distinct events. Only large fragments containing the entire membrane spanning sequence of OmpA are able to assemble into a conformation exhibiting native resistance to proteolytic digestion (Klose et al., 1988a).
In general, amino acid substitutions or insertions within outer membrane loops exposed on the cell surface are well tolerated and do not interfere with the folding of the protein in the membrane. Peptides as large as 60 amino acids have been inserted within external loops of various outer membrane proteins and appear to be exposed on the surface of intact E. coli cells as indicated by immunochemical techniques (Charbit et al., 1991). However, efforts to direct soluble reporter proteins such as alkaline phosphatase, to the cell surface using outer membrane protein fragments have not been successful. These fusions either end up at incorrect cellular locations or become anchored in the membrane with the secreted protein domain facing the periplasm (Murphy et al., 1990). In gram-negative bacteria the outer membrane acts as a barrier to restrict the export of proteins from the cell. Normally only pilins, flagellins, specific enzymes and a few toxins are completely transported across the outer membrane (Kornacker and Pugsley, 1990). Most of these proteins are first secreted into the periplasmic space via the general secretion pathway and then cross the outer membrane by a process that involves the action of several additional gene products (Filloux et al., 1990).
Whole cell adsorbents are considered to have potential value for biotechnology applications for the purification of various molecules or the selective removal of hazardous compounds from contaminated waste waters. However, a major constraint in the development of whole cell adsorbents is the availability of bacterial strains with suitable ligands on their surface. Although functional antibody fragments have been produced in Escherichia coli (Skerra and Pluckthun 1988, Better et al. 1988, Orlandi et al. 1989, Sastry et al. 1989), these polypeptides have not been expressed on the cell surface. Indeed, a "library" of recombinant immunoglobulins containing both heavy and light variable domains (Huse et al. 1989) has been produced with the proteins having antigen-binding affinity comparable to the corresponding natural antibodies. Furthermore, the variety of recombinant immunoglobulins from bacteria is greater than the number of antibody molecules that can be generated by the mammalian cell. In this way it has become possible to expand the repertoire of antibodies that can be made by the immune system (Huse et al. 1989). While the availability of such a wide range of immunoglobulins suggests the potential for creation of E. coli cells endowed with immunological surface receptors, there has been little success in producing recombinant proteins on the surface of bacterial cells, and conspicuous lack of a method to generate recombinant immunoglobulins on surfaces of gram negative host cells.
Although the potential repertoire of immunoglobulins produced in an immunized animal is high (&gt;10.sup.10), only a small number of monoclonal antibodies can be generated using hybridomas. This limitation complicates the isolation of antibodies with specific properties, such as the ability to act as a catalyst. Combinational antibody libraries comprising millions of genes of different antibodies have been cloned using phage .lambda.(Huse et al., 1989). However, screening the library to select the desired clone can be extremely time consuming and complicated. One approach to the screening problem has been an attempt to express antibodies on the surface of filamentous phage. Phage particles displaying high affinity antibody molecules on their surface can be enriched by chromatography through a column of immobilized antigen (Barbas et al., 1991; Clarckson et al., 1991; Breitling, 1991). Although the feasibility of this technique has been demonstrated, several problems are apparent, including: (1) fusion to bacteriophage coat proteins causing interference with antibody folding, (2) subcloning of large numbers of positive phage particles in order to produce soluble antibody fragments to carry out more extensive characterization, and (3) lack of control of the number of antibody molecules on the phage surface, thus affecting binding to the immobilized antigen and complicating the selection procedure.